We then tested new-generation DNA polymerases that are designed to have increased performance relative to Taq DNA polymerase.įor our experiments, we focused on a particular genomic region and a set of 25 plant samples (Supplementary Table S1) that we knew contained mononucleotide repeats that negatively impacted sequence quality. We first attempted to reduce the degree of stutter generated with Taq DNA polymerase–mediated PCR by increasing the affinity of the polymerase by varying the PCR conditions. Here we report our efforts designed to improve the quality of sequence data generated from PCR-amplified genomic regions containing mononucleotide repeats. Kieleczawa ( 16) reported that sequences containing A/T repeats up to 50 bp can be readily sequenced from plasmids, indicating that slippage during the sequencing reaction is not the main source of reduced sequence quality. Slipped-strand mispairing does seem to be primarily (although not wholly) a function of the PCR process. Determining the nucleotide sequence between these regions is usually impossible to do with any confidence without the added step of designing internal sequencing primers ( 16). Confounding the problem further are samples that have two or more regions of mononucleotide repeats within targets of interest.
While contigs can usually be generated by sequencing both strands, this necessitates doubling the amount of sequencing effort required in order to obtain a minimum 2-fold coverage. In particular, A/T rich regions such as intergenic spacers of the plastid genome-widely used by investigators of plant phylogenetics ( 15)-often contain mononucleotide repeats. This issue is regularly encountered by those who sequence PCR products derived from genomic DNA. Additionally, the quality of sequence data after a mononucleotide repeat of 10 or more bases is often greatly reduced, often to the point of being unreadable. These mutations can result in stutter products in microsatellite images or in sequence chromatograms, and may confound the delimitation of the true repeat number, as stutter products can be generated in similar or even greater proportions than the true product ( 9, 14).
SEQUENCHER COMPARE CHROMATOGRAMS FULL
It is assumed that when the active site of a polymerase is full of identical nucleotides, it is more likely to dissociate ( 9) and allow the DNA strands an opportunity to misalign. They occur at the greatest rate in mononucleotide repeats (also known as homopolymer runs) composed of eight or more nucleotides ( 9), which is the estimated number of bases that fill the active site of Taq DNA polymerase and many other DNA polymerases ( 11–13). In PCR, these artifacts of the process have a greater mutation frequency with greater repeat numbers, and smaller sizes of repeat motifs ( 8–10). Deletion mutations are believed to be more common as they require fewer nucleotides of the dsDNA to dissociate and therefore are more energetically favorable than insertion mutations ( 5–7). If base pairing is disrupted after polymerase dissociation, then a loop of one or more repeat units may form in either the nascent or the template strand prior to re-association and cause the insertion or deletion of one or more units, respectively, in the newly formed DNA strand ( 1, 2). In order for slipped-strand mispairing to occur, the DNA polymerase enzyme first stalls and dissociates from the dsDNA complex during replication of the repeated motif. Mutations at SSR sites during in vitro enzymatic replication of SSRs are usually the result of insertion or deletion of repeats in the extending, or nascent, DNA strand sequence ( 5). The natural process of slipped-strand mispairing that results in SSR mutation in vivo also occurs in vitro during PCR-mediated DNA replication.
The relatively rapid rate of mutation, high number of alleles, and their high frequency in genomes have made SSRs popular markers for population genetics, linkage mapping, genetic fingerprinting, and taxonomic study ( 3, 4). These highly repetitive motifs make microsatellites particularly prone to mutation via slipped-strand mispairing ( 1, 2). Simple sequence repeats (SSRs), or micro-satellites, are repetitive nucleotide sequences composed of 1–6 bases found in both organellar and nuclear genomes.
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